| Because of extremely low breeding rate,population quantity of the giant panda become less.Although artificial breeding technology has already obtained notable achievements,the reproductive problems such as repeated infertility also exist on the whole.Enormous number and different species of microbes plant in vagina,which plays an important role in the health of the body.The bacterial or fungal vaginosis is a kind of vaginal disorders caused by microflora,which may be associated with numerous adverse consequences such as premature delivery and the other related infectious diseases.In particular,the occurrence and development of diseases have inseparable connection with the composition and abundance of microbial flora.There are many factors of reproductive disorders in giant pandas.We attempted to investigate the vaginal microbes in the giant pandas with cultured and uncultured methods,to understand their population and abundance,and to lay the foundation for the role of microbes of reproductive disorders in the giant pandas.Vaginal microbial species and their predominant strains in vaginas from 21 giant pandas were conducted with 454 high-throughput sequencing and bioinformatics analysis methods,meanwhile,the biological characteristics of cuturable fungal population and high abundance fungi in vaginas of giant pandas was studied.The results showed that:(1)Total 224 110 16 S rRNA optimized sequences were measured in vaginal samples from 21 giant pandas,which were divided into 6 650 operational taxonomic units(OTUs),in which belonged to 2 147 species,618 genera from 23 bacterial phyla.Among them,however,there were 184 species,136 genera from 3 phyla not to be identified.The predominant species was confirmed at each classfication levels.The predominant phyla identified were:Firmicutes(54.44%),Proteobacteria(39.13%),Bacteroidetes(5.70%)and Actinobacteria(4.07%).The most commonly predominant genera were:Streptococcus(21.14%),Lactococcus(13.63%),Pseudomonas(11.62%),Enterococcus(6.64%)and Ralstonia(6.27%),The predominate species were:Streptococcus_gallolyticus_subsp._pasteurianus(14.28%)and Lactococcus piscium(11.79%)etc.(2)Galactococcus(13.63%),Enterococcus(6.64%)and lactobacillus(0.01%)were the microbes that mainly produce lactate in vaginas of the giant pandas,in which Galactococcus was the most predominant one,but lactobacillus was not because it only accounted for 0.01%of total number of bacteria sequences.(3)Total 155 480 18 S rRNA optimized eukaryotic sequences were detected in vaginal samples from 20 giant pandas.Among these,there were 1 650 OTUs,which contained 92 genera and 14 phyla from 2 eukaryote kingdom.And these belonged to 34 species animals(27 species insects),70 species fungi and 27 species plants.The animal sequences account for 98.1%of the total number of sequences,and the predominant phyla identified were:multicellular animals(96.4%),platyhelminthes(1.28%)and Rotatoria(0.13%),respectively.Total 13 genera could be identified to genus levels,and the predominant ones were:Malassezia(1.26%),Alternaria(0.53%),Prototheca(0.07%),Candida(0.06%),Pichia((0.01%),Guehomyces(0.01%)and Trichosporon(0.01%),respectively.The predominant parasites were:Paraschneideria,Colpoda,Gregarina,Cryptosporidium,Bodomorpha,Cercomonas,Entamoeba,Vannella and Spumella.But there were partial sequences of animals,fungi and plants not to be identified,and needs to be studied further.(4)Through culture,isolation and identification based on morphology and molecular biology of the fungi from 10 vaginal secretions samples,total 30 species were identified,which contained 25 molds(83.33%)and 5 yeasts(16.67).(5)The biological characterictic of Alternaria spp.strain isolated from the giant panda vagina was investigated in this experiment.The results showed that the culture medium which was the first suitable for its culture was CMA and AA,the second was PDA,SDA and CZA,and the third was RGA(it was the most unsuitable for growth).The optimal growth temperature for this fungus was at 25 T,poor growth was at 15?,20? and 30?,but almost no growth was in environment at 35?.The optimal pH for Alternaria spp was pH6 and pH7,poor growth was pH8-pH11,the worst was the pH4 and pH5.Most suitable carbon source for its growth was starch and maltose,followed by glucose,sucrose and glycerol,and the worst was lactose.The most suitable nitrogen source to the fungal growth was sodium nitrate,followed by ammonium oxalate,poor growth was urea,almost no growth was ammonium sulphate.(6)The biological characteristic of Candida catenulate strain isolated from vaginas in the giant pandas was conducted in this study.The results showed that C.catenulate thallus was elliptical,grow branching pseudohyphae and true hyphae in corn twain medium and its spore was not thick-walled.In the other respects,it had no-logarithmic growth curve in the growth phase,segmented and no germ tube in serum incubation.Glucose,galactose and ethanol assimilation tests were positive;trehalose,cellobiose,raffinose and inositol assimilation tests were negative;maltose and galactose fermentation tests positive;glucose,lactose,inulin and sucrose fermentation tests negative;the urea test was negative;the resistance of actidione test was positive.Colour reaction test was green.In this study,we understood more comprehensively the vaginal population,abundance and diversity of bacteria and eukaryotes in the giant pandas.We obtained 30 species fungi and completed the survey on biological characteristics of main dominant fungi through the traditional methods of isolation and culture.These results vigorously expand provious sporadic reports about the vaginal culturable microorganism in the giant pandas,fill the gaps that know well vaginal microbe diversity in the giant pandas,and also provide basic data for improving breeding rate of giant pandas. |
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